help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tsuchida, T.
Right arrow Articles by Fujita, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tsuchida, T.
Right arrow Articles by Fujita, T.
Endocrinology Vol. 140, No. 2 865-870
Copyright © 1999 by The Endocrine Society

ARTICLES

Inhibition of Stimulated Amylase Secretion by Adrenomedullin in Rat Pancreatic Acini

Tomohiro Tsuchida, Hirohide Ohnishi, Yuji Tanaka, Tetsuya Mine and Toshiro Fujita

Fourth Department of Internal Medicine, University of Tokyo School of Medicine, Tokyo 112-8688, Japan

Address all correspondence and requests for reprints to: Dr. Hirohide Ohnishi, M.D., Ph.D., Fourth Department of Internal Medicine, University of Tokyo School of Medicine, 3–28-6 Mejirodai, Bunkyo-ku, Tokyo 112-8688, Japan. E-mail: hohnishi-tky{at}umin.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adrenomedullin is a novel hypotensive peptide originally isolated from human pheochromocytoma and recently localized to PP cells of the pancreatic islets of Langerhans. Based on the pancreatic islet-acinar axis model, we investigated the effect of adrenomedullin on regulated exocytosis of exocrine pancreas. Using rat [125I]adrenomedullin, specific binding sites were localized to rat pancreatic acini. We next examined the effect of adrenomedullin on 100 pM cholecystokinin (CCK)-stimulated amylase release from pancreatic acini. Adrenomedullin inhibited amylase secretion in a dose-dependent manner by approximately 50% at maximum, and the IC50 was 1.1 pM. However, adrenomedullin did not affect rat [125I]CCK binding to isolated acini or reduce the intracellular free Ca2+ concentration increased by CCK. Adrenomedullin also inhibited amylase secretion induced by 1 µM calcium ionophore A23187, suggesting that adrenomedullin inhibits stimulated amylase secretion by functioning at a step(s) distal to the ligand-receptor binding system and intracellular calcium mobilizing mechanism. In streptolysin-O permeabilized acini, 10 nM adrenomedullin shifted the calcium dose-response curve to the right, indicating that adrenomedullin inhibits calcium-induced amylase secretion by reducing calcium sensitivity of the pancreatic exocytotic machinery. In addition, pretreatment of pancreatic acini with pertussis toxin abolished the inhibitory effect of adrenomedullin on CCK-stimulated amylase secretion. These results indicate that adrenomedullin inhibits stimulated amylase secretion by reducing the calcium sensitivity of the exocytotic machinery of the pancreatic acini. A pertussis toxin-sensitive GTP-binding protein(s) is also involved in this mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADRENOMEDULLIN is a novel hypotensive peptide, first purified from a human adrenal pheochromocytoma (1). It is expressed mainly in cardiovascular tissue, including heart, aorta, and kidney, as well as adrenal medulla (2). When human adrenomedullin was administered iv into rats, it caused a rapid and marked reduction of blood pressure, along with the decrease in peripheral vascular resistance. Thus, adrenomedullin is assumed to be an important hormone, participating in the regulation of cardiovascular function (3, 4). In addition to its cardiovascular action, various extravascular actions of adrenomedullin have recently been described. Adrenomedullin has been shown to modify the proliferation of mesangial cells (5) and fibroblasts (6). Adrenomedullin has also been shown to modulate regulated secretion from various organs, such as adrenal gland (7), pituitary gland (8), and stomach (9). More recently, immunoreactive adrenomedullin has been localized to various extracardiovascular organs (10), including PP cells of pancreatic islets of Langerhans (11). In PP cells, moreover, adrenomedullin has been revealed to be colocalized with PP in secretory granules by immunoelectron microscopy. In addition, whereas exogenous adrenomedullin inhibited insulin secretion from isolated rat pancreatic islet, the monoclonal antiadrenomedullin antibody (which neutralized adrenomedullin bioactivity) was able to increase insulin release from isolated pancreatic islets, in the absence of exogenous adrenomedullin (11). These data suggest the secretion of adrenomedullin from PP cells of pancreatic islets and the regulation of insulin secretion by adrenomedullin in a paracrine manner.

The pancreas consists of exocrine acini and endocrine islets. Between the two constituents, there exists the pancreatic islet-acinar axis. Namely, because all vascular flow is directed from the pancreatic islets to the acini, various islet hormones are assumed to modulate acinar secretion in a paracrine manner (12). For example, insulin from islet B-cells has been shown to enhance cholecystokinin (CCK)-stimulated amylase release from pancreatic acini (13). In contrast, SRIF from islet D-cells has been shown to inhibit pancreatic acinar secretion triggered by the combination of CCK and secretin (14). Given that adrenomedullin is present in secretory granules of pancreatic islet PP-cells and might be released from them, we hypothesize that adrenomedullin may have an effect on pancreatic acinar exocytosis. Therefore, the present study was conducted to study the action of adrenomedullin on the regulated exocytosis of pancreatic acini and to elucidate its mechanism. To this end, we first determined the presence of adrenomedullin receptor on pancreatic acini. We next studied the effect of adrenomedullin on CCK-stimulated amylase release from pancreatic acini. Experiments are then expanded using [125I]CCK binding assay, intracellular free calcium and cAMP measurements, and streptolysin-O permeabilized acinar technique to gain insights into the ligand-receptor system and the intracellular events influenced by adrenomedullin. In addition, we determined the role of a pertussis toxin (PTX)-sensitive GTP-binding protein (G protein) in the action of adrenomedullin. Our results indicate that adrenomedullin inhibits regulated exocytosis of pancreatic acini by reducing the calcium sensitivity of the exocytotic machinery via its specific receptor, and that a PTX-sensitive G protein is also involved in this regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Collagenase (type CLISPA) was purchased from Worthington Biochemical Corp. (Freehold, NJ). Soybean trypsin inhibitor (SBTI), BSA, 3-isobutyl-1-methyl-xanthine (IBMX), A23187, and ATP (Mg-ATP) were purchased from Sigma Chemical Co. (St. Louis, MO). Rat adrenomedullin, CCK octapeptide (CCK-8, sulfate), rat {alpha} calcitonin gene-related peptide (CGRP), human secretin, and SRIF (SS-14) were purchased from Peptide Institute (Osaka, Japan). PTX was purchased from Seikagaku Corp. Kogyo (Tokyo, Japan), [125I]adrenomedullin from Amersham (Tokyo, Japan), [125I]CCK-8 (sulfate) from DuPont (Boston, MA), streptolysin-O from Murex (Dartford, England), and Fura-2 and pluronic F-127 from Molecular Probes, Inc. (Eugene, OR).

Preparation of isolated pancreatic acini
Pancreatic acini were prepared from the pancreata of 170- to 200-g male Wistar rats, by the collagenase digestion method, as previously described (15). Isolated acini were suspended in HEPES-buffered Ringer’s solution (HRB) containing 10 mM HEPES/NaOH (pH 7.4), 118 mM NaCl, 4.7 mM KCl, 1 mM Na2HPO4, 1.13 mM MgCl2, 5.5 mM D-glucose, 2.5 mM CaCl2, Eagle’s MEM amino acid supplement, 2 mM L-glutamine, 0.2% BSA, and 0.01% SBTI. After a 30-min recovery incubation, the following experiments were carried out.

Receptor binding assays
For the adrenomedullin receptor binding assay, isolated intact acini were incubated for 5 min at 30 C in HRB containing 1 kBq rat [125I]adrenomedullin and various concentrations of unlabeled adrenomedullin or CGRP. Bound and free [125I]adrenomedullin were separated by microcentrifugation, followed by washing three times with 1 ml cold PBS. The binding of [125I]adrenomedullin to pancreatic acini was measured by liquid scintillation counting. Nonspecific binding was determined in the presence of 1 µM unlabeled adrenomedullin. Specific binding was defined as total binding minus nonspecific binding (nonspecific binding: 22.5 ± 3.5% of total binding). Binding data were analyzed by nonlinear regression using the Stat View program (Abacus Concepts, Berkeley, CA) to calculate the dissociation constant and the maximum concentration of binding. The CCK receptor binding assay was carried out using [125I]CCK and various concentrations of unlabeled adrenomedullin or CCK by the same method as described above.

Measurement of amylase release from intact acini
Isolated acini were suspended in HRB and incubated in a shaking water bath at 37 C for 30 min with 100 pM CCK or 1 µM calcium ionophore A23187, in the presence or absence of adrenomedullin. Amylase released into the supernatant during incubation was quantified using the Phadebas Amylase Test (Dai-ichi Pure Chemicals, Tokyo, Japan) and expressed as the percent of total amylase in the acini at the beginning of the incubation.

Measurement of intracellular cAMP concentration
For the measurement of cAMP concentration in pancreatic acini, enzymatically isolated acini were suspended in HRB and incubated with the appropriate agents for 30 min at 37 C in the presence of 0.1 mM IBMX. The incubated acini were spun down and lysed in 200 µl 0.25% dodecyltrimethylammonium bromide, followed by microcentrifugation at top speed for 10 sec. Using 100-µl samples of the supernatant, cAMP assay was carried out with cAMP assay kit (Amersham, Buckinghamshire, UK), according to the protocol of the manufacturer.

Measurement of intracellular free calcium concentration ([Ca2+]i)
Intact isolated acini were incubated with 2 µM fura-2 AM and 0.04% F-127 at ambient temperature for 30 min and then washed and resuspended in HRB. A 2-ml aliquot of fura-2-loaded acini was used for the determination of [Ca2+]i using a digital imaging system (Hitachi F-2000, Hitachi, Tokyo, Japan). The sample was excited at 340 nm and 380 nm, and emission was monitored at 510 nm. Because it is impossible to calculate absolute [Ca2+]i from experiments performed in this manner, the ratio Em 340 nm/Em 380 nm was used as a relative index of [Ca2+]i instead. The [Ca2+]i in acini exposed to 100 pM CCK was compared in the presence and absence of 10 nM adrenomedullin.

Measurement of amylase release from streptolysin-O permeabilized acini
Isolated intact acini were suspended in permeabilization buffer consisting of 20 mM piperazine diethanesulfonic acid (pH 7.0), 140 mM potassium glutamate, 0.91 mM MgCl2, 5 mM EGTA, 1 mg/ml BSA, 0.1 mg/ml SBTI, 1 mM Mg-ATP, and 0.5 IU/ml streptolysin-O, and aliquoted into 500-µl samples. In the presence or absence of 10 nM adrenomedullin, amylase release was initiated by adding 500 µl permeabilization buffer supplemented with CaCl2, to give various concentrations of free Ca2+, which were calculated using a computer program as described previously (15). Amylase release was quantified by the same method as described above.

Preparation of PTX-treated pancreatic acini
PTX-treated acini were obtained by injecting 50 µg of the toxin into the peritoneal cavity of intact rats, as previously described (16), where we have shown that PTX well catalyzed ADP-ribosylation of G protein in pancreatic acini prepared by this method. Seventy-two hours after the toxin injection, pancreatic acini were isolated, and 100 pM CCK-stimulated amylase release, in the presence of various concentrations of adrenomedullin, was examined as described above. Because it has been previously demonstrated that the PTX-pretreatment of pancreatic acini performed in this manner does not affect the CCK-stimulated intracellular signal-transducing system (including intracellular calcium mobilization and inositol phosphate accumulation) (17) or does not alter stimulated or basal amylase secretion from pancreatic acini (16), the effect of PTX pretreatment on adrenomedullin inhibitory action on CCK-stimulated amylase secretion can be examined in this system.

Statistical analysis
Statistical analysis was performed using the two-way layout ANOVA, unless otherwise indicated. P < 0.05 was considered sig- nificant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adrenomedullin receptor on rat pancreatic acini
We first examined whether a specific adrenomedullin receptor is present on rat pancreatic acini. Because adrenomedullin has been shown to share a common receptor with CGRP in some cell systems (18, 19, 20), competitive binding assays were carried out with CGRP, as well as adrenomedullin. Competitive binding curves of [125I]adrenomedullin to isolated acini, in the presence of unlabeled rat adrenomedullin or rat CGRP, are shown in Fig. 1Go. Binding of [125I]adrenomedullin to isolated acini was inhibited by unlabeled adrenomedullin but not by CGRP. These results demonstrate that specific adrenomedullin receptor is present on pancreatic acini. The dissociation constant and the maximum concentration of binding values of the receptor were 38 pM and 10 fmol/mg protein, respectively.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 1. Competition curves of [125I]adrenomedullin binding to rat pancreatic acini. Acini were incubated at 30 C for 5 min with various concentrations of unlabeled adrenomedullin ({blacksquare}) or unlabeled CGRP (•). Values are the means ± SE for three independent experiments using separate acinar preparations obtained from three rats, with assays performed in triplicate.

 
Inhibitory effect of adrenomedullin on CCK-stimulated amylase release from pancreatic acini
To examine whether adrenomedullin has an effect on regulated exocytosis of exocrine pancreas, we investigated its effect on CCK-stimulated amylase release from pancreatic acini. As shown in Fig. 2Go, adrenomedullin inhibited 100 pM CCK-stimulated amylase release in a dose-dependent manner. Maximum inhibition was 52%, observed at 10 nM adrenomedullin. The half-maximum inhibition was observed at 1.1 pM adrenomedullin. However, adrenomedullin did not inhibit or enhance basal amylase secretion, even at 10 nM. These results demonstrate that adrenomedullin specifically attenuates stimulated amylase release from pancreatic acini.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 2. Effect of adrenomedullin on CCK-stimulated amylase secretion. Acini were incubated for 30 min with various concentrations of adrenomedullin in the presence ({blacksquare}) and absence (•) of 100 pM CCK. Values are the means ± SE for three independent experiments using separate acinar preparations obtained from three rats, with assays performed in triplicate. *, P < 0.01, compared with CCK-stimulated amylase secretion, in the absence of adrenomedullin.

 
Effect of adrenomedullin on [125I]CCK binding to isolated acini
In an attempt to elucidate the point of action of the adrenomedullin inhibitory effect on stimulated amylase release, we first examined the effect of adrenomedullin on the binding rate of [125I]CCK to isolated acini. As shown in Fig. 3Go, whereas unlabeled CCK inhibited the binding of [125I]CCK to CCK receptor, unlabeled adrenomedullin did not affect the binding of [125I]CCK to CCK receptor. These results indicate that adrenomedullin is not a CCK receptor antagonist and that its inhibitory effect on amylase secretion acts at a point distal to the ligand-receptor binding of CCK.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 3. Effect of adrenomedullin on [125I]CCK binding to rat pancreatic acini. Acini were incubated at 30 C for 5 min with various concentrations of unlabeled adrenomedullin ({blacksquare}) or unlabeled CCK (•). Values are the means ± SE for three independent experiments using separate acinar preparations obtained from three rats, with assays performed in triplicate.

 
Effect of adrenomedullin on intracellular cAMP concentration in pancreatic acinar cells
In other tissues, adrenomedullin has been shown to exert its physiological effect by modulating intracellular cAMP concentration in target cells (5, 6, 18). To assess the involvement of the intracellular cAMP mobilizing system in the inhibitory action of adrenomedullin in regulated exocytosis of pancreatic acini, we examined the effect of adrenomedullin on intracellular cAMP concentration in pancreatic acinar cells. We used secretin and SRIF as positive controls of intracellular cAMP-mobilizing hormones (14). As shown in Table 1Go, although secretin sufficiently increased intracellular cAMP concentration and SRIF reduced the secretin-enhanced cAMP concentration in pancreatic acinar cells, adrenomedullin did not alter the intracellular cAMP concentration in the presence or absence of CCK and did not reduce the secretin-enhanced cAMP concentration in pancreatic acinar cells. These data indicate that adrenomedullin inhibits stimulated-amylase secretion by mechanisms other than intracellular cAMP mobilization.


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of adrenomedullin on intracellular cAMP concentration in pancreatic acinar cells

 
Effect of adrenomedullin on [Ca2+]i Increased by CCK
To further characterize the point of the action of adrenomedullin distal to ligand-receptor binding of CCK, we examined the effect of adrenomedullin on the CCK-induced increase in [Ca2+]i. As shown in Fig. 4Go, 100 pM CCK caused the [Ca2+]i to increase in isolated pancreatic acini (A), however, 10 nM adrenomedullin failed to reduce this CCK-induced increase in [Ca2+]i (B). In addition, 10 nM adrenomedullin did not affect the [Ca2+]i in pancreatic acini at resting state (B). These data imply that the inhibitory effect of adrenomedullin on amylase secretion acts at a point distal to the receptor-mediated intracellular calcium mobilization.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 4. Effect of adrenomedullin on [Ca2+]i increase induced by CCK. [Ca2+]i was measured using fura-2 loaded pancreatic acini. Values are the ratio Em 340 nm/Em 380 nm. A, Pancreatic acini were stimulated with 100 pM CCK; B, pancreatic acini were treated with 10 nM adrenomedullin, 1 min before the stimulation by 100 pM CCK. The results are representative of four independent experiments using four separate acinar preparations obtained from four rats, with at least three calcium determinations.

 
Inhibitory effect of adrenomedullin on amylase secretion induced by calcium ionophore A23187
To demonstrate directly that adrenomedullin inhibits stimulated amylase secretion at a point distal to the receptor-mediated intracellular calcium mobilizing system, we examined the effect of adrenomedullin on calcium ionophore A23187-stimulated amylase release. Because A23187 raises intracellular calcium by bypassing the receptor-mediated second-messenger generation, we can assess the effect of adrenomedullin on events distal to the intracellular calcium mobilization. As shown in Fig. 5Go, adrenomedullin inhibited amylase secretion stimulated by 1 µM A23187 in a dose-dependent manner. Maximum inhibition was observed at 100 nM adrenomedullin. This result suggests that adrenomedullin inhibits stimulated amylase secretion from pancreatic acini by acting at a step that comes after intracellular calcium mobilization.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 5. Effect of adrenomedullin on A23187-stimulated amylase secretion. Acini were incubated for 30 min with 1 µM A23187 and various concentrations of adrenomedullin. Values are the means ± SE for four independent experiments using separate acinar preparations obtained from four rats, with assays in triplicate. *, P < 0.01; **, P < 0.05, compared with A23187-stimulated amylase release in the absence of adrenomedullin. Basal amylase release was 2.9 ± 0.2% of total.

 
Inhibitory effect of adrenomedullin on amylase release from streptolysin-O permeabilized acini
To further characterize the inhibitory mechanism of adrenomedullin, we measured amylase secretion from permeabilized acini. Figure 6Go shows that amylase secretion, in the absence of adrenomedullin, was stimulated by calcium in a dose-dependent manner and that maximum amylase release was observed at 1 µM Ca2+. In the presence of adrenomedullin, however, calcium-induced amylase secretion decreased at all calcium concentrations. Adrenomedullin reduces amylase secretion by shifting the dose-response curve for calcium sensitivity to the right. These results indicate that adrenomedullin inhibits stimulated amylase secretion by reducing intracellular calcium sensitivity.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 6. Effect of adrenomedullin on calcium-stimulated amylase secretion from streptolysin-O permeabilized acini. Permeabilized acini were incubated for 5 min with various concentrations of free calcium in the presence ({blacksquare}) and absence (•) of 10 nM adrenomedullin. The result is representative of four independent experiments using separate acinar preparation obtained from four rats, with assays in triplicate.

 
Effect of PTX on adrenomedullin-mediated inhibition of amylase secretion
To determine whether a G protein(s) is involved in the adrenomedullin-mediated inhibition of amylase secretion from pancreatic acini, we investigated the effect of PTX on such inhibition. When pancreatic acini were pretreated with PTX, the inhibitory effect of adrenomedullin on CCK-stimulated amylase secretion was completely abolished (Fig. 7Go). This result demonstrates that the inhibitory action of adrenomedullin on amylase secretion from pancreatic acini is mediated by a PTX-sensitive G protein(s).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 7. Effect of adrenomedullin on CCK-stimulated amylase secretion from PTX-treated acini. Acini were prepared from rats pretreated with PTX. Acini were incubated for 30 min with 100 pM CCK and various concentrations of adrenomedullin. Values are the means ± SE for three independent experiments using separate acinar preparations obtained from three PTX-pretreated rats, with assays performed in triplicate. Basal amylase release from PTX-pretreated acini was 3.1 ± 0.3% of total.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although adrenomedullin has been localized to PP cells of pancreatic islet (11) and is supposed to have some action on pancreatic acini, based on pancreatic islet-acinar axis theory, an effect of adrenomedullin on pancreatic acinar function has not been previously demonstrated. In this study, we have described the action of adrenomedullin on regulated exocytosis of pancreatic acini. First, we showed that an adrenomedullin-specific receptor is present on pancreatic acini. We next showed that adrenomedullin attenuated CCK- stimulated amylase release from pancreatic acini without affecting CCK binding to its receptor, intracellular cAMP concentration, or intracellular free calcium concentration. We further demonstrated that adrenomedullin inhibits A23187-stimulated amylase release and shifted the calcium dose-response curve of permeabilized acini to the right, which suggests that adrenomedullin inhibits stimulated-amylase secretion by reducing the calcium sensitivity of exocytotic machinery of pancreatic acini. We also demonstrated that a pertussis-toxin sensitive G protein(s) is involved in the mechanism of adrenomedullin inhibition of amylase secretion from pancreatic acini.

To date, only two peptides have been shown to inhibit stimulated amylase secretion in vitro: SRIF and epidermal growth factor (EGF). SRIF attenuates cAMP-enhanced amylase secretion by inhibiting the cAMP-dependent signaling pathway in pancreatic acini (14, 16), whereas EGF inhibits amylase secretion stimulated by calcium-mobilizing secretagogues by inhibiting secretagogue-induced phospholipase C activation (21, 22). Although adrenomedullin also inhibits amylase secretion induced by the calcium-mobilizing secretagogue CCK, the inhibitory mechanism is distinct from that of EGF. Because adrenomedullin does not reduce the CCK-induced increase in intracellular free calcium concentration, it is unlikely that adrenomedullin inhibits phospholipase C activation. Thus, adrenomedullin is a potent inhibitor of calcium-stimulated pancreatic acinar exocytosis, with the novel mechanism that attenuating the calcium sensitivity of exocytotic machinery.

Adrenomedullin has previously been reported to inhibit stimulated exocytosis in various tissues. For example, adrenomedullin inhibited CRH-stimulated ACTH secretion from rat anterior pituitary cells (8) and glucose-stimulated insulin secretion from pancreatic islets of Langerhans (11). However, the intracellular mechanism of these adrenomedullin inhibitory actions had not been addressed. The results of this study, then, expand these previous studies by showing the mechanism of adrenomedullin inhibitory action. Moreover, the inhibitory mechanism of adrenomedullin that we presented here is unique. Adrenomedullin has been demonstrated to show its action on various cellular functions by enhancing the intracellular cAMP production of target cells through both receptors specific to adrenomedullin and those shared with CGRP (23, 24). Recently, an adrenomedullin-specific receptor has been cloned from rat lung and shown to mediate cAMP-dependent signaling (25). Although adrenomedullin binds to a specific receptor in pancreatic acini, as shown in Fig. 1Go, we assume that the adrenomedullin receptor on pancreatic acini might be distinct from the cloned one because of the lack of mediating intracellular cAMP production. In addition to intracellular cAMP production, adrenomedullin has been revealed to stimulate intracellular free calcium mobilization through its specific receptor in endothelial cells (24). In contrast, however, adrenomedullin did not mobilize intracellular free calcium in pancreatic acini, as shown in Fig. 4Go. Thus, the adrenomedullin receptor on pancreatic acini might also be distinct from that on endothelial cells. Consistent with our suggestion, it has been recently reported that multiple isoforms of adrenomedullin-specific receptor may exist (20). Therefore, it is reasonable to speculate that the mechanism of the inhibitory action of adrenomedullin on stimulated amylase secretion proposed here might involve signal transduction via a adrenomedullin-specific receptor distinct from those mediate cAMP production and intracellular free calcium mobilization in other cell systems.

A new observation made in this study is that the inhibitory effect of adrenomedullin in pancreas via the specific receptor is mediated by a PTX-sensitive G protein. Previous studies have shown that a cholera toxin-sensitive G protein is involved in the adrenomedullin signaling pathway (24). From our observation, however, we cannot determine whether or not the PTX-sensitive G protein is coupled to the adrenomedullin receptor on pancreatic acini. Indeed, there still remains the possibility that the PTX-sensitive G protein involved in the adrenomedullin signaling pathway in pancreas might be present on an intracellular organelle, because multiple heterotrimeric G proteins have been localized to various intracellular organelles, such as zymogen granules and the trans-golgi network in pancreatic acinar cells (26, 27, 28). In any case, cloning of the adrenomedullin receptor on pancreatic acinar cells is necessary for a further understanding of the mechanism of the inhibitory action of adrenomedullin on the regulated exocytosis of pancreatic acini.

In summary, we have elucidated an inhibitory action of adrenomedullin on regulated exocytosis of exocrine pancreas. The mechanism of the inhibition operates distal to the intracellular calcium mobilization, which modulates the sensitivity of exocytotic machinery to cytosolic free calcium via an adrenomedullin-specific receptor and a PTX-sensitive G protein. For further understanding of the physiological action of adrenomedullin on exocrine pancreas, in vivo studies are necessary to investigate the effect of adrenomedullin on amylase secretion and the regulation of adrenomedullin secretion from pancreatic islets.

Received June 29, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T 1993 Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 192:553–560[CrossRef][Medline]
  2. Ichiki Y, Kitamura K, Kangawa K, Kawamoto M, Matsuo H, Eto T 1994 Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Lett 338:6–10[CrossRef][Medline]
  3. Ishiyama Y, Kitamura K, Ichiki Y, Nakamura S, Kida O, Kangawa K, Eto T 1993 Hemodynamic effects of a novel hypotensive peptide, human adrenomedullin, in rats. Eur J Pharmacol 241:271–273[CrossRef][Medline]
  4. Sakata J, Shimokubo T, Kitamura K, Nakamura S, Kangawa K, Matsuo H, Eto T 1993 Molecular cloning and biological activities of rat adrenomedullin, a hypotensive peptide. Biochem Biophys Res Commun 195:921–927[CrossRef][Medline]
  5. Chini EN, Choi E, Grande JP, Burnett JC, Dousa TP 1995 Adrenomedullin suppresses mitogenesis in rat mesangial cells via cAMP pathway. Biochem Biophys Res Commun 215:868–873[CrossRef][Medline]
  6. Withers DJ, Coppock HA, Seufferlein T, Smith DM, Bloom SR, Rozengurt E 1996 Adrenomedullin stimulates DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 cells. FEBS Lett 378:83–87[CrossRef][Medline]
  7. Yamaguchi T, Baba K, Doi Y, Yano K 1995 Effect of adrenomedullin on aldosterone secretion by dispersed rat adrenal zona glomerulosa cells. Life Sci 56:379–387[Medline]
  8. Samson WK, Murphy T, Schell DA 1995 A novel vasoactive peptide, adrenomedullin, inhibits pituitary ACTH release. Endocrinology 136:2349–2352[Abstract]
  9. Rossowski WJ, Jiang NY, Coy DH 1997 Adrenomedullin, amylin, calcitonin gene-related peptide and their fragments are potent inhibitors of gastric acid secretion in rats. Eur J Pharmacol 336:51–63[CrossRef][Medline]
  10. Ichiki Y, Kitamura K, Kangawa K, Kawamoto M, Matsuo H, Eto T 1995 Distribution and characterization of immunoreactive adrenomedullin in porcine tissue, and isolation of adrenomedullin [26–52] and adrenomedullin [34–52] from porcine duodenum. J Biochem (Tokyo) 118:765–770[Abstract/Free Full Text]
  11. Martinez A, Weaver C, Lopez J, Bhathena SJ, Elsasser TH, Miller MJ, Moody TW, Unsworth EJ, Cuttitta F 1996 Regulation of insulin secretion and blood glucose metabolism by adrenomedullin. Endocrinology 137:2626–2632[Abstract]
  12. Williams JA, Goldfine ID 1985 The insulin-pancreatic acinar axis. Diabetes 34:980–986[Abstract]
  13. Matsushita K, Okabayashi Y, Koide M, Hasegawa H, Otsuki M, Kasuga M 1994 Potentiating effect of insulin on exocrine secretory function in isolated rat pancreatic acini. Gastroenterology 106:200–206[Medline]
  14. Matsushita K, Okabayashi Y, Hasegawa H, Koide M, Kido Y, Okutani T, Sugimoto Y, Kasuga M 1993 In vitro inhibitory effect of somatostatin on secretin action in exocrine pancreas of rats. Gastroenterology 104:1146–1152[Medline]
  15. Burnham DB, Williams JA 1982 Effects of carbachol, cholecystokinin, and insulin on protein phosphorylation in isolated pancreatic acini. J Biol Chem 257:10523–10528[Abstract/Free Full Text]
  16. Ohnishi H, Mine T, Kojima I 1994 Inhibition by somatostatin of amylase secretion induced by calcium and cyclic AMP in rat pancreatic acini. Biochem J 304:531–536
  17. Matozaki M, Sakamoto C, Nagao M, Nishizaki H, Baba S 1998 G protein in stimulation of PI hydrolysis by CCK in isolated rat pancreatic acinar cells. Am J Physiol 255:E652–E659
  18. Eguchi S, Hirata Y, Kano H, Sato K, Watanabe Y, Watanabe TX, Nakajima K, Sakakibara S, Marumo F 1994 Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells. FEBS Lett 340:226–230[CrossRef][Medline]
  19. Hall JM, Siney L, Lippton H, Hyman A, Jaw KC, Brain SD 1995 Interaction of human adrenomedullin13–52 with calcitonin gene-related peptide receptors in the microvasculature of the rat and hamster. Br J Pharmacol 114:592–597[Medline]
  20. Owji AA, Smith DM, Coppock HA, Morgan DGA, Bhogal R, Ghatei MA, Bloom SR 1995 An abundant and specific binding site for the novel vasodilator adrenomedullin in the rat. Endocrinology 136:2127–2134[Abstract]
  21. Stryjek-kaminska D, Piper A, Caspary WF, Zeuzem S 1995 Epidermal growth factor inhibits hormone- and fibroblast growth factor-induced activation of phospholipase C in rat pancreatic acini. Peptides 16:123–128[CrossRef][Medline]
  22. Piper A, Stryjek-kaminska D, Klengel R, Zeuzem S 1997 Epidermal growth factor inhibits bombesin-induced activation of phospholipase C-ß1 in rat pancreatic acinar cells. Gastroenterology 113:1747–1755[CrossRef][Medline]
  23. Ishizaka Y, Ishizaka Y, Tanaka M, Kitamura K, Kangawa K, Minamino N, Matsuo H, Eto T 1994 Adrenomedullin stimulates cyclic AMP formation in rat vascular smooth muscle cells. Biochem Biophys Res Commun 200:642–646[CrossRef][Medline]
  24. Shimekake Y, Nagata K, Ohta S, Kambayashi Y, Teraoka H, Kitamura K, Eto T, Kangawa K, Matsuo H 1995 Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic endothelial cells. J Biol Chem 270:4412–4417[Abstract/Free Full Text]
  25. Kapas S, Catt KJ, Clark AJL 1995 Cloning and expression of cDNA encoding a rat adrenomedullin receptor. J Biol Chem 270:25344–25347[Abstract/Free Full Text]
  26. Denker SP, McCaffery JM, Palade GE, Insel PA, Farquhar MG 1996 Differential distribution of {alpha}-subunits and ß[{gamma}]-subunits of heterotrimeric G proteins on golgi membranes of the exocrine pancreas. J Cell Biol 133:1027–1040[Abstract/Free Full Text]
  27. Ohnishi H, Ernst SA, Yule DI, Baker CW, Williams JA 1997 Heterotrimeric G protein Gq/11 localized on pancreatic zymogen granules is involved in calcium-regulated amylase secretion. J Biol Chem 272:16056–16061[Abstract/Free Full Text]
  28. Padfield PJ, Panesar N 1997 Identification of Go{alpha}, Gq{alpha}, and Gs{alpha} Immunoreactivity associated with the rat pancreatic zymogen granule membrane. Biochem Biophys Res Commun 237:235–238AU: Please define "NS".[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tsuchida, T.
Right arrow Articles by Fujita, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tsuchida, T.
Right arrow Articles by Fujita, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals